ABSTRACT: The one-carbon metabolism pathway is strongly implicated in the development of hepatocellular carcinoma (HCC) in patients with non-alcoholic fatty liver disease (NAFLD). The incidence of HCC has increased in the US, and about a third of all HCC cases diagnosed in the US do not have a history of viral hepatitis or alcohol abuse, but these cases of HCC often have clinical or biochemical features of NAFLD. Indeed, NAFLD-related HCC cases are expected to increase further in the US. Functionally, the one-carbon metabolism pathway regulates DNA methylation through metabolism of one-carbon nutrients, such as choline, methionine and folate, to form S-adenosylmethionine (SAM). SAM is the universal methyl donor used for DNA methylation; the formation of 5-methylcytosine (5mC). Following the transfer of methyl groups from SAM for methylation, SAM is converted to S-adenosylhomocysteine (SAH), which is a potent inhibitor of the activities of DNA methyltransferases (DNMTs) and thus suppresses DNA methylation. Hence, while SAM promotes methylation, SAH inhibits methylation, and the SAM/SAH ratio reflects a ?hepatic methylation index? because both SAM and SAH are formed primarily in the liver. Additionally, recent data show that 5mC is not a static DNA mark, it is converted to 5-hydroxymethylcytosine (5hmC) by TET enzymes. 5hmC is now recognized as a unique epigenetic modification to DNA that is distinct from 5mC. However, both 5mC and 5hmC are involved in gene regulation and both have been implicated in the progression of established HCC, but it is unclear which of these DNA marks is most relevant to HCC development. The central hypothesis of this proposal is that individual differences in hepatic methylation index or epigenetic modifications to DNA influence susceptibility to HCC in isolation or in concert with genetic variants in one-carbon, DNMT or TET genes. The Specific Aims are: (1) to identify common and rare genetic risk variants in the one-carbon metabolism pathway associated with HCC risk. (2) To assess the association between hepatic methylation index (SAM/SAH ratio) and HCC risk, and identify one-carbon gene modifiers of the association. (3) To assess the association between epigenetic modifications to DNA (5mC and 5hmC) and HCC risk, and evaluate interaction by one-carbon, DNMT, and TET genes. Findings from this research are expected to yield novel insights into the one-carbon metabolism pathway as a previously under-recognized, potentially modifiable contributor to HCC and improve strategies for the prevention, risk stratification, and early detection of HCC. The activities outlined in this application are designed to extend the applicant?s expertise in genetic and molecular epidemiology, and acquire new skills in epigenetics, metabolomics, and bioinformatics. Members of the mentoring team have complementary expertise that spans these disciplines. Overall, the cross-disciplinary, integrative molecular epidemiologic approach of this proposal and the career development activities that include formal coursework and ?hands-on? workshops will enable the applicant to acquire the needed competencies to foster his transition to independence.